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J. Biol. Chem., Vol. 276, Issue 36, 33504-33511, September 7, 2001

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A Novel Nuclear Human Poly(A) Polymerase (PAP), PAP^*

Christina B. Kyriakopoulou, Helena Nordvarg^§, and Anders Virtanen^¶

From the  Department of Cell and Molecular Biology, Uppsala University, Box 596, Uppsala SE-75124 and the ^§ Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala SE-75185, Sweden

Received for publication, May 21, 2001, and in revised form, June 21, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Poly(A) polymerase (PAP) is present in multiple forms in mammalian cells and tissues. Here we show that the 90-kDa isoform is the product of the gene PAPOLG, which is distinct from the previously identified genes for poly(A) polymerases. The 90-kDa isoform is referred to as human PAP (hsPAP). hsPAP shares 60% identity to human PAPII (hsPAPII) at the amino acid level. hsPAP exhibits fundamental properties of a bona fide poly(A) polymerase, specificity for ATP, and cleavage and polyadenylation specificity factor/hexanucleotide-dependent polyadenylation activity. The catalytic parameters indicate similar catalytic efficiency to that of hsPAPII. Mutational analysis and sequence comparison revealed that hsPAP and hsPAPII have similar organization of structural and functional domains. hsPAP contains a U1A protein-interacting region in its C terminus, and PAP activity can be inhibited, as hsPAPII, by the U1A protein. hsPAP is restricted to the nucleus as revealed by in situ staining and by transfection experiments. Based on this and previous studies, it is obvious that multiple isoforms of PAP are generated by three distinct mechanisms: gene duplication, alternative RNA processing, and post-translational modification. The exclusive nuclear localization of hsPAP establishes that multiple forms of PAP are unevenly distributed in the cell, implying specialized roles for the various isoforms.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The majority of mammalian mRNAs end with a 200- to 250-adenosine residue tail at their 3'-ends. The function of the poly(A) tail is not fully understood, but studies have highlighted its role in regulating gene expression via translation and mRNA stability (1-4). The mRNA poly(A) tail is added post-transcriptionally, and the biochemistry of mammalian nuclear polyadenylation has been extensively studied (reviewed in Refs. 5-8). Polyadenylation is a multistep and multicomponent reaction and proceeds through two separable steps, pre-mRNA cleavage and adenosine addition. Both reactions are dependent on a highly conserved sequence element, the hexanucleotide AAUAAA. At least six trans-acting protein factors are required for the reaction in vitro (5-8).

Poly(A) polymerase (PAP),¹ is the enzyme responsible for mRNA poly(A) tail synthesis. PAP has been identified and cloned from several eukaryotic species, e.g. yeast, human, mouse, bovine, frog, and chicken (9-19). Interestingly, multiple forms of PAP are present in cell lines and tissues of several species (9, 11, 13-16, 19-21). In HeLa cell nuclear extracts, three isoforms, having apparent molecular masses of 90, 100, and 106 kDa, have been found (16). The molecular mechanisms for generating all these isoforms are still not completely understood. However, molecular cloning has established that at least five isoforms of full-length PAP can be generated by alternative RNA processing (15, 17).² It is also known that phosphorylation contributes to the multiplicity of PAP (9, 16, 20). Recently it has been established that PAP and PAP-related genes are present in the human genome (11, 13, 14, 22). Therefore, so far at least three distinct mechanisms can generate multiple isoforms of PAP: gene duplication, alternative RNA processing, and post-translational modification. These phenomena unexpectedly increase the diversity of PAP and raise questions about the functional significance of multiple PAPs in vivo.

It seems reasonable to hypothesize that different PAPs are responsible for different functions in vivo, because PAP participates in a whole set of different reactions, e.g. RNA cleavage at the poly(A) site and AAUAAA-dependent or -independent poly(A) tail synthesis (23, 24). Biochemical fractionation studies have indicated that different forms of PAP reside at different subcellular compartments (16, 25). A testis-specific PAP has been identified, suggesting that some isoforms of PAP are restricted to certain developmental stages or tissues (11, 13).

In this report we have molecularly cloned the human 90-kDa PAP isoform, previously identified in HeLa nuclear extracts. The 90-kDa isoform is encoded by a distinct locus recently identified as a PAP-related gene (22). The gene has been named PAPOLG, and its product is hsPAP. In a recent report (14) the same gene was implicated in monoadenylation of small RNAs. Here, we show that hsPAP is a bona fide poly(A) polymerase harboring both nonspecific and CPSF/AAUAAA-dependent polyadenylation activity. hsPAP is exclusively localized in the nucleus.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plasmid Constructs-- Full-length hsPAP and various deletion mutants were molecularly cloned by a standard RT-PCR procedure using the following strategy. A 371-amino acid N-terminal fragment of hsPAP was amplified by RT-PCR using HeLa total RNA and primers a and b, subcloned into pGEM-T vector (Promega Inc.), and further subcloned into the pET-32(a) vector (Novagen Inc.) between the NcoI and SacI sites. The resulting clone was called pPAP(H1-371), where H denotes the N-terminal tag and the numbers refer to amino acids in full-length hsPAP. C-terminal deletions of hsPAP were cloned by inserting PCR products derived with primer pairs c-d, c-e, c-f, and c-g between the EcoRI and SacI sites of pPAP(H1-371) giving rise to pPAP(H1-493), pPAP(H1-506), pPAP(H1-575), and pPAP(H1-683), respectively. The C terminus of hsPAP was identified and cloned into pGEM-T vector by 3'-RACE using the CLONTECH Smart Race cDNA amplification kit and gene-specific primers h and i according to the manufacturer. After identification of the C-terminal end, including the stop codon, primer pair c-j was used for generation of pPAP(H1-736) as described above. An N-terminal deletion mutant was amplified using primer pair k-j for deletion of the first 16 amino acids and the resulting clone was named pPAP(H17-736). The clone pPAP(H521-683) was generated as outlined above using primers l and g. The pET-32(a) vector introduces an N-terminal thioredoxin-tag which increases the expression of the soluble recombinant protein, a histidine-tag and an S-tag enabling easy purification via affinity chromatography. Full-length hsPAP and one C-terminal deletion mutant were also subcloned into the pCAL-c vector (Stratagene) between the NcoI and KpnI sites, using the same strategy as above (primer pairs a-j and a-g) giving rise to pPAP(1-736C) and pPAP(1-683C), respectively. Full-length hsPAP and C-terminal deletion mutants were also subcloned into the EGFP-C2 vector (CLONTECH) between the XhoI and KpnI sites using primer pairs m-b, m-e, and m-j. The resulting clones were named pPAP(EGFP1-371), pPAP(EGFP1-506), and pPAP(EGFP1-736), respectively. Primers used were as follows: (a) 5'-CACCATGGAAGAGATGTCTGCAAACACC-3'; (b) 5'-GAGAGCTCTTAGGTACCCCTATACTTTTGAAAGAAATTCGGTGG-3'; (c) 5'-GCCTGTCTGGGATCCTCGGGT-3'; (d) 5'-GAGAGCTCTTAGGTACCGTGAAGTTGTTTTTTCTTTACATGAGTTGC-3'; (e) 5'-GAGAGCTCTTAGGTACCCTTTTTCTTCTTTTGAAGAATTTCTGC-3'; (f) 5'-GAGAGCTCTTAGGTACCACTCAGTGGCTTCTCCACAATTACA-3'; (g) 5'-GAGAGCTCTTAGGTACCTTTTCTTTTTCTTTCTTCAGCAGTGCG-3'; (h) 5'-CAACACCTCACAACCCTGCCCA-3'; (i) 5'-GAGATCCCATTCCCCATCCATAG-3'; (j) 5'-GAGAGGTACCAAGCCGATTAAGGGTCAGTCG-3'; (k) 5'-CACCATGGGAAAGCATTATGGAATTACCTC-3'; (l) 5'-CACCATGGAATCCAAAAGATTGTCTCTGGATAGC-3'; and (m) 5'-CACACTCGAGGCAATGGAAGAGATGTCTGCAAAC-3'. Restriction sites for cloning are included in the primer sequences shown in boldface: NcoI in a; KpnI and SacI in b, d, e, f, and g and XhoI in m. A stop codon was introduced between the KpnI and SacI sites. The KpnI restriction site was introduced to enable cloning into the pCALc vector and adds two extra amino acids at the C terminus of all the pET-32a clones expressing hsPAP. The NcoI cloning site in primer a introduces a point mutation at the second amino acid in the sequence changing lysine to glutamate. All clones have been sequenced using the Big-Dye Terminator sequencing kit (Applied Biosystems).

Buffers Used for Purification of Recombinant Proteins-- Buffer A (20 mM Hepes/KOH, pH 7.5, 0.5 M KCl, 1.0% Nonidet P-40, 1.0% Tween-20, 10% glycerol, 5 mM imidazole, 20 mM -mercaptoethanol (b-MEOH)), buffer B (buffer A omitting detergents and b-MEOH), buffer C (20 mM Hepes/KOH, pH 7.5, 0.5 M KCl, 10% glycerol, 200 mM imidazole), buffer D (buffer A containing 50 mM imidazole), buffer E (buffer D omitting detergents and b-MEOH and containing 0.05 M KCl), buffer F (buffer E containing 200 mM imidazole), buffer G (20 mM Hepes/KOH, pH 8.8, 0.05 M KCl, 10% glycerol, 0.5 mM DTT, 1.5 mM MgCl₂), buffer H (buffer G containing 0.5 M KCl), buffer I (50 mM Tris/HCl, pH 7.5, 0.15 M KCl, 0.1% Triton X-100, 10% glycerol, 1 mM Mg, 2 mM CaCl₂, 1 mM imidazole, 10 mM b-MeOH), buffer J (buffer I containing 0.2 M KCl), buffer K (buffer I omitting b-MEOH, containing 0.25 M KCl), buffer L (50 mM Tris/HCl, pH 7.5, 1 M KCl, 2 mM CaCl₂, 2 mM EDTA, 0.5 mM DTT, 1.5 mM MgCl₂).

Expression and Purification of Recombinant Forms of PAP-- Expression plasmids were used to transform BL21(DE3) pLysS Escherichia coli strains. One colony was used for inoculation of 50-100 ml of TB medium in the presence of 50 µg/ml carbenicillin and 34 µg/ml chloramphenicol and grown overnight at 37 °C without shaking. The 50- to 100-ml culture was inoculated into a final 0.5- to 1-liter culture in TB medium containing antibiotics. Bacteria were grown at 37 °C (vigorous shaking) and were induced with 0.42 mM isopropyl-1-thio--D-galactopyranoside plus 0.524 mM MgCl₂ at A₆₀₀ ~ 0.5-1.0. Cells were harvested by centrifugation 3 h post-induction, and pellets were frozen at 70 °C. Extracts for His-tagged PAP were prepared by unthawing the cells on ice and lysing with buffer A containing 1 tablet of EDTA-free protease inhibitors), followed by sonication (three times 10 s), centrifugation 20 min at 39,000 × g, and 0.45-µm filtration. Extracts were mixed with 1 ml of Talon metal affinity resin (CLONTECH) equilibrated in buffer A, and proteins were bound batch-wise by 1-h rotation. The resin was washed with buffer A and subsequently washed with buffer B, and the proteins were eluted with buffer C. The eluate was loaded onto a HiTrap chelating column (Amersham Pharmacia Biotech) equilibrated with buffer D. The column was washed with buffer D and subsequently with buffer E. Proteins were eluted with buffer F. The eluate was loaded on a Heparin HiTrap column equilibrated in buffer G, washed with the same buffer and proteins were eluted with buffer H. Extracts for calmodulin-tagged PAP where prepared as described above, but cells were lysed in buffer I containing 1 tablet of EDTA-free protease inhibitors. Extracts were mixed batch-wise with 0.75 ml of calmodulin affinity resin (Stratagene) equilibrated in buffer I, and proteins were bound overnight. The resin was washed with buffer J followed by buffer K. Proteins were eluted with buffer L. Protease inhibitors 0.5 mM phenylmethylsulfonyl fluoride, 1.0 µg/ml leupeptin, 1.0 µg/ml pepstatin, and 1.0 µg/ml aprotinin were added freshly to all buffer solutions, and all procedures were performed at 4 °C.

Antibodies-- Polyclonal antiserum specific for the C-terminal of hsPAP were generated by immunizing two rabbits using 0.45 mg/rabbit recombinant purified PAP(H521-683) polypeptide, followed by three boost injections with the same amount of antigen. The sera was named anti-PAP. Peptide antiserum specific for PAPII was purchased from Sigma-Genosys using a synthetic peptide (N-terminal: CKTSSTDLSDIPA) corresponding to amino acids 715-726 of hsPAPII for immunization, and was named anti-PAPIIex22. Monoclonal antibodies were Y12 (26) and 20:14 (16).

SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis-- HeLa nuclear extracts were purchased from the Computer Cell Culture Center. SDS-polyacrylamide gel electrophoresis was carried out according to a previous study (27) as was Western blotting (28). Detection was done by anti-mouse or anti-rabbit immunoglobulin, horseradish peroxidase-linked whole antibody (Amersham Pharmacia Biotech) diluted 1:1000, and ECL plus chemiluminescence reagent (Amersham Pharmacia Biotech).

Poly(A) Polymerase Assay Conditions-- Nonspecific polyadenylation activity assays were carried out as described previously (21, 29) with modifications optimizing the activity; the reaction mixture (25 µl) contained: 100 mM Tris/HCl buffer, pH 8.6 (measured at room temperature), 40 mM KCl, 0.040 mM EDTA, 10% glycerol, 1 mM DTT, 9 units of RNasin (ribonuclease inhibitor), 0.1% Nonidet P-40, 0.5 mM MnCl₂, 0.5 mg/ml bovine serum albumin, 0.5 mM cold ATP, 1.2 µCi of [-³²P]ATP (3000 Ci/mmol) and 2 µM oligoA₁₅ (Dharmacon), and the reaction was performed for 20 min at 37 °C. One unit of PAP is defined as the amount of enzyme needed for incorporation of 1 pmol of AMP per min. Reaction rate was measured in a linear range versus PAP concentrations (8-23 nM) and time (10-30 min). Kinetic parameters were determined using oligoA₁₅ in the concentration range 0.0125-2 µM for the full-length hsPAP and hsPAPII. In the case of kinetic estimations for the deletion mutants the same enzyme concentration was used, however, the primer concentration was in the 0.5-5 times K_m range. Unthawed recombinant hsPAP and hsPAPII were stabilized by addition of 0.05% Nonidet P-40, 20% glycerol, and 1 mM DTT for the time kept on ice. Reactions were stopped by precipitation of the insoluble polyadenylated product in acid conditions (5% trichloroacetic acid-1% sodium pyrophosphate) in glass fiber filters and washed three times with 5% trichloroacetic acid (30).

The U1A inhibition assay of hsPAP was done using the U1A di-peptide (from N termini to N termini; CAAAERDRKREKRKAAAA(K)AAAAKRKERKRDREAAAC where (K) is the branched lysine) kindly provided by Dr. S. Gunderson, or control U1A mono-peptide (CAAAERDRKREKRKAAAA, Sigma-Genosys) in the nonspecific polyadenylation assay described above but modified to conditions previously described (31, 32). The reaction mixture above was modified to final concentrations: 20 mM Tris/HCl, 60 mM KCl, 10% glycerol, 5 mM DTT, 0.1-0.2 µM oligoA₁₅, and no peptide, 9.6 pmol of U1A di-peptide or U1A mono-peptide, respectively.

The specific polyadenylation activity was carried out as described previously (29, 33) with modifications to normalize the differences in between the specific and nonspecific assays in this study. ³²P-Labeled and capped RNA substrates (L3(54), L3G(54)) were synthesized by in vitro transcription and purified as previously described (34). CPSF partially purified from calf thymus (35) and recombinant hsPAP were used as specified in the figure legends. The reaction mixture (25 µl) contained: 100 mM Tris/HCl buffer, pH 8.3 (measured at room temperature), 40 mM KCl, 0.040 mM EDTA, 9.6% glycerol, 1 mM DTT, 9 units of RNasin (ribonuclease inhibitor), 0.01% Nonidet P-40, 0.72 mM MgCl₂, 1 mM ATP, 2.5% polyvinylalcohol, 20 mM creatinine phosphate, and the reaction was performed for 20 min at 30 °C. The reaction was stopped in Proteinase K buffer, and the incubated RNA product was extracted and resolved in 10% polyacrylamide (acrylamide/bisacrylamide 19:1)-7 M urea.

Immunocytochemical Methods-- HeLa cells were grown up to 50-70% confluency on coverslips in the presence of Dulbecco's modified Eagle's medium supplemented with glutamine and 10% fetal calf serum (Life Technologies, Inc.). Coverslips were washed two times in PBS and fixed in 1% paraformaldehyde (PFA) in PBS (pH 7.3) for 3 min, extracted with 0.5% Triton X-100 in PBS for 15 min, and then post-fixed in 4% PFA in PBS for 10 min. For immunofluorescence staining the following antibodies were used: Primary antibodies; 20:14, anti-PAP, Y12, and anti-PAPIIex22 in dilutions 1:2, 1:40, 1:10, 1:20, respectively; the respective pre-immune serum was used in the cases of polyclonal antibodies at the same dilutions. Secondary antibodies; species-specific goat anti-mouse IgG coupled to biotin (Amersham Pharmacia) or to Alexa Fluor 488 (emission at green spectrum) (Molecular Probes), species-specific goat anti-rabbit IgG coupled to Alexa Fluor 594 (emission at red spectrum) (Molecular Probes). Fixed cells were incubated for 30 min with blocking reagent buffer (5% Eliza blocking reagent, Roche Molecular Biochemicals) at room temperature. Subsequently they were incubated for 30 min or 2 h with primary antibodies diluted in blocking reagent and washed 3 × 5 min in PBS. Secondary antibodies were incubated for 30 min or 1 h and washed 3 × 5 min in PBS. In the case where dual staining experiments were performed with monoclonal antibody 20:14 and anti-PAP and a biotin labeled anti-mouse secondary antibody was used, the cells were washed 4 × 5 min in PBS and incubated for 1 h with streptavidin coupled to Alexa Fluor 488 (Molecular Probes). All coverslips were mounted in Vectashield (Vector Laboratories) and shielded. Where polyclonal serum was used, a control pre-immune serum was used to subtract the background signal. Fluorescence microscopy was performed in an Axioplan 2 imaging fluorescence microscope, using a 100× objective lens. Image analysis was done by the Axion vision.3 software.

Transfection Methods-- HeLa cells, grown up to 50% confluency, were transfected using plasmids pPAP(EGFP1-371), pPAP(EGFP1-506), and pPAP(EGFP1-736), and left to grow for 10 or 24 h. The Superfect transfection reagent (Qiagen) was used, and conditions were optimized for ratio of DNA:transfection reagent, as suggested by the manufacturer. Cells were fixed in 4% PFA (in PBS, pH 7.3), shielded, and analyzed in a fluorescence microscope by excitation at 495 nm and emission at the green spectrum.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Molecular Cloning of Human PAP-- Monoclonal antibodies NN:2 and 20:14 raised against hsPAPII recognize three isoforms of PAP: 90, 100, and 106 kDa in sizes (16). However, a polyclonal antibody raised against bovine PAPII recognizes only the two larger forms (24, 36). A reason for this discrepancy could be that the monoclonal antibodies recognize a common epitope shared among all three isoforms of PAP, whereas the polyclonal antibody is directed against epitopes not present in the 90-kDa form. This experimental evidence suggests that the 90-kDa isoform of PAP has unique antigenic epitopes unrelated to the 100- and 106-kDa forms, implying that the 90-kDa isoform could be encoded by a separate gene.

To identify potential human PAP-related genes we regularly searched using the BLAST algorithm (37) high throughput (htgs) and non-redundant sequence data bases, while they were being released during the human genome sequencing project (38). During these searches we identified a PAP-related sequence in the human genomic clone (AC011245.6) located on chromosome 2. The same locus has recently been identified as a PAP-related gene and as a small RNA monoadenylating enzyme (14, 22). Further data base searches revealed several overlapping expressed sequence tags. These results combined with 3'-RACE semi-nested RT-PCR allowed us to predict the sequence of an mRNA encoding a potential PAP. The novel human gene was named PAPOLG and its product hsPAP. The sequence information was used to molecularly clone cDNAs originating from HeLa cells by RT-PCR. A schematic drawing of the exon/intron organization of hsPAP and comparison to the previously reported gene hsPAPII is shown in Fig. 1A. The deduced amino acid sequence of hsPAP is presented in Fig. 1B and compared with the bovine and human PAPII. Structural and functional domains/motifs are also represented. A comparison using the ClustalX algorithmic approach (39) showed that hsPAP has an overall identity of 67% at the nucleotide level and 60% identity at the amino acid level.

View larger version (47K): [in this window] [in a new window]   Fig. 1.   A, exon organization of the human PAP and PAPII genes. A schematic drawing of the exon organization of hsPAP and hsPAPII genes are shown. Relative sizes of exons (boxes) are indicated whereas the real sizes of introns (thin lines) have not been included for simplicity. Boxes above the thin lines indicate the identity at the nucleotide level whereas boxes below the thin lines indicate identity at the amino acid level. Black, gray, and open boxes refer to degree of identity in percentage as outlined. The number of nucleotides (nt) and amino acids (aa) for hsPAP are: exons 1 (17 nt, 6 aa), 2 (162, 54), 3 (67, 22), 4 (82, 27), 5 (110, 37), 6 (54, 18), 7 (112, 37), 8 (90, 30), 9 (139, 47), 10 (73, 24), 11 (121, 40), 12 (85, 29), 13 (54, 18), 14 (120, 40), 15 (110, 36), 16 (122, 41), 17 (161, 54), 18 (89, 29), 19 (221, 74), 20 (66, 22), 21 (57, 19), 22 (96, 32). The number of nucleotides (nt) and amino acids (aa) for hsPAPII are: exons 1 (8 nt, 3 aa), 2 (174, 58), 3 (67, 22), 4 (82, 27), 5 (110, 37), 6 (54, 18), 7 (112, 37), 8 (90, 30), 9 (139, 47), 10 (73, 24), 11 (121, 40), 12 (85, 29), 13 (54, 18), 14 (120, 40), 15 (110, 36), 16 (122, 41), 17 (143, 48), 18 (101, 33), 19 (239, 80), 20 (63, 21), 21 (75, 25), 22 (93, 32). B, comparison of hsPAP, hsPAPII, and bovine PAPII. The deduced amino acid sequence of hsPAP (AC011245.6, AC012498), is compared with hsPAPII (P51003) and bovine PAPII (P25500). Stars indicate identical amino acids in all three sequences. Structural domains are denoted on the basis of the resolved crystal structure of bovine PAPII (43). Bars and arrows represent alpha helices and beta strands, respectively. The catalytic domain is shown in gray, the central domain is in black and the C-terminal RNA binding domain in light gray. Open boxes show the location of NLS 1, NLS 2, a putative NLS 3 element in hsPAP, a cyclin recognition motif and the U1A-interacting region. Amino acids important for catalytic function are indicated in boldface.

Organization of the hsPAP Gene-- The genes encoding hsPAPII and hsPAP span 62.5 and 37 kb of genomic sequences, respectively. They both contain 22 exons, and all splice sites obey the GT/AG rule (40). The topology and the sizes of exons 2-16 are shared between the two genes, implicating that they share a common ancestor and arose through gene duplication (41, 42). Sequence comparison (Fig. 1A) revealed that the exons 1 of both genes were unrelated to each other; exons 2-16 were highly conserved both at the amino acid and nucleotide levels, having an overall identity of ~75% at both levels; exons 17-21 were less conserved in their sequences whereas exon 22 exhibits a high degree of identity both at the amino acid and nucleotide levels. The last half of exon 22 encodes a potential U1A protein-interacting region (see also below).

Structural Organization of hsPAP-- An inspection of known structural and functional motifs/domains in hsPAPII revealed that several of those were conserved in hsPAP. These motifs/domains included amino acids important for catalysis, recognition of the ATP substrate, and RNA binding (29, 43) (Fig. 1B). The cyclin-recognition motif and four of the seven consensus and non-consensus phosphorylation sites that have been mapped for cyclin dependent kinases were conserved (44, 45). Two nuclear localization signals (NLS) (46) were conserved between the two PAPs, whereas a third putative bipartite NLS was found in the C-terminal end of hsPAP. The sequence encompassing the U1A protein interaction region is highly conserved (14 out of 18 amino acids).

The 90-kDa Isoform Is the Product of the Novel hsPAP Gene-- To raise an antiserum specific for hsPAP, we molecularly cloned the C-terminal region of hsPAP spanning amino acids 521-683 into the pET32(a) vector. The recombinant polypeptide was expressed in E. coli, purified to homogeneity, and used to raise an hsPAP-specific antiserum, named anti-PAP. Fig. 2A shows that the obtained serum was specific for hsPAP, because it only recognized recombinant versions of hsPAP and not hsPAPII. In these experiments C-terminally calmodulin-tagged recombinant proteins were used to exclude recognition of the N-terminal-located tags present in the polypeptide used for immunization. As predicted the monoclonal antibody (20:14) raised against hsPAPII recognized hsPAP (Fig. 2B). An analysis of C-terminal deletion constructs of hsPAP revealed that the epitope was located in the highly conserved N-terminal region of hsPAP and hsPAP II (Figs. 1B and 2B)

View larger version (66K): [in this window] [in a new window]   Fig. 2.   The 90-kDa isoform of human PAPs is PAP. A, C-terminally calmodulin-tagged recombinant proteins expressed and purified from E. coli were resolved by 6.25% SDS/polyacrylamide electrophoresis, blotted, and subsequently probed with anti-PAP polyclonal serum (lanes 1-4, dilution 1:4000) or with preimmune serum (lanes 5-8). Lanes 1 and 5, PAP(1-736C); lanes 2 and 6, PAP(1-683C); lanes 3 and 7, PAPII(1-745C); lanes 4 and 8, protein purified from E. coli containing the calmodulin vector only. B, recombinant proteins purified from E. coli were resolved by 7% SDS/polyacrylamide electrophoresis, blotted, and probed with 20:14 monoclonal antibody (dilution 1:20). Lane 1, PAPII (H1-745); lane 2, PAP(H1-683); lane 3, PAP(H1-575); lane 4, PAP(H1-506); lane 5, PAP(H1-493); and lane 6, PAP(H1-371). C, 2000 µg of HeLa nuclear extracts were loaded in a 6-cm-wide lane, resolved by 6% SDS/polyacrylamide electrophoresis, blotted, and transferred to an Immobilon-P membrane. The membrane was cut into 0.5-cm wide strips, and each strip was probed with different antibodies. Lane 1, 20:14; lanes 2 and 3, anti-PAP diluted 1:2000 and 1:4000, respectively; lane 4, preimmune serum diluted 1:2000; lane 5, anti-PAPIIex22 polyclonal serum diluted 1:1000; lane 6, preimmune serum diluted 1:1000; lane 7, 20:14. The position of molecular size markers are indicated in kilodaltons.

To investigate if the anti-PAP serum recognized the 90-kDa isoform of HeLa cell PAP, we probed HeLa nuclear extracts. Fig. 2C shows that the serum exclusively recognized the 90-kDa species. An hsPAPII-specific polyclonal antiserum, named anti-PAPIIex22, was raised against a synthetic peptide of exon 22 (amino acids 715-726) of hsPAPII. An affinity-purified anti-PAPIIex22 recognized the 100- and 106-kDa mobility species and not the 90-kDa isoform (Fig. 2C). Thus, we conclude that the 90-kDa isoform corresponds to hsPAP, the product of the PAPOLG gene.

Properties of hsPAP-- To investigate if hsPAP had polyadenylating activity, we used the nonspecific polyadenylation assay in the presence of Mn(II) and various nucleotide tri-phosphates. The assay was designed so that the amount of hsPAP was provided in excess. Fig. 3A shows that recombinant PAP(H1-736) exhibited specificity for incorporation of ATP, whereas incorporation of UTP, GTP, CTP, and dATP were inefficient and stopped after the addition of one to two molecules of the respective nucleotide analogue.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   hsPAP is a bona fide poly(A) polymerase. A, specificity for ATP. Polyadenylation activity assays in the presence of Mn(II) were performed as detailed under "Experimental Procedures." Ribonucleotides and dATP, as indicated, were tested at 0.5 mM. 5'-End-labeled primer oligoA15 (300 fmol), and recombinant PAP(H1-736) (300 fmol (lanes 2, 5, 8, 11, 14), 600 fmol (lanes 3, 6, 9, 12, 15), or 900 fmol (lanes 4, 7, 10, 13, 16)) were added to the reactions. The ratio of enzyme to primer was 3:1. Lane 1, no PAP added; lanes 2-4, ATP; lanes 5-7, UTP; lanes 8-10, GTP; lanes 11-13, CTP; and lanes 14-16, dATP. Reactions were incubated at 37 °C for 30 min, and reacted oligoA15 was purified and resolved in a 16% sequencing polyacrylamide (acrylamide:bis, 19:1)-7 M urea gel. The resulting gel was exposed and analyzed by a 400S PhosphorImager (Molecular Dynamics). S and P denote location of oligoA15 substrate and polyadenylated product, respectively. B, hsPAP exhibits CPSF/AAUAAA-dependent polyadenylation activity. Specific polyadenylation activity was performed as detailed under "Experimental Procedures." The reaction contained 70 fmol of RNA substrate (L3(54) in lanes 1-10 and L3G(54) in lanes 11 and 12), CPSF, PAP(H1-736), and/or PAP(H1-371) were included as indicated. Lane 3, 130 fmol of PAP(H1-371); lanes 4-11, 20, 40, 80, 160, 320, 640, 80, and 80 fmol of PAP(H1-736), respectively. The incubated RNA substrates were extracted as described in panel A and resolved in 10% polyacrylamide-7 M urea gel.

PAPII acquires specificity for hexanucleotide containing mRNAs in the presence of cleavage and polyadenylation specificity factor (CPSF) (33). To investigate whether hsPAP had this classical property, we performed specific polyadenylation assays in the presence of Mg(II) and partially purified CPSF from calf thymus (35). Fig. 3B shows that hsPAP exhibited CPSF/AAUAAA-dependent activity, because the L3(54) RNA substrate was efficiently polyadenylated compared with the hexanucleotide mutated L3G(54) RNA substrate. Furthermore, hsPAP did not exhibit any polyadenylation activity in the presence of Mg(II) when CPSF was omitted. A C-terminal deletion mutant PAP(H1-371) was inactive, as described for bovine PAPII (29).

We conclude that hsPAP exhibits the fundamental catalytic properties for a bona fide poly(A) polymerase, i.e. specific incorporation of ATP- and CPSF/AAUAAA-dependent polyadenylation activity.

Kinetic Parameters and Mutational Analysis of hsPAP-- To further characterize hsPAP we determined the kinetic parameters (K_m and V_max). Nonspecific polyadenylation activity was measured in the presence of Mn(II) and highly purified oligoA₁₅. The K_m for hsPAP was shown to be 0.051 µM using a variety of calculation methods. The ratio V_max/K_m that represents the real efficiency of the reaction in terms of affinity to the primer and actual catalytic capacity was determined. The kinetic parameters for hsPAP (Table I) are in a similar range as for recombinant hsPAPII and for the reported bovine PAPII (24, 29). A calmodulin tag at the C terminus of hsPAP and hsPAPII did not significantly alter the kinetic parameters (Table I). Thus, recombinant hsPAP has similar kinetic properties as hsPAPII.

Functional Analysis of C- and N-terminal Deletions Mutants-- To identify regions in hsPAP important for polyadenylation activity, we constructed C-terminal deletion mutants (see "Experimental Procedures"). In Table I the K_m and V_max/K_m for these deletion mutants are shown. The increased K_m of PAP(H1-493) and PAP(H1-506) compared with full-length PAP(H1-736) suggests that hsPAP contains a primer binding domain spanning the NLS 1 region, as proposed for mammalian PAPII (29). Interaction with CPSF has previously been implicated in this region of bovine PAPII (29). Table II shows the nonspecific and specific polyadenylation activities of deletion mutants and full-length hsPAP using the same molar amounts of recombinant polypeptides throughout the comparison. In the nonspecific assay, C-terminal deletions up to amino acid 575 retained 100% of the activity when compared with the full-length PAP(H1-736). Truncation up to the C-terminal end of NLS 1 (PAP(H1-506)) led to a remaining activity of 65% whereas further deletion of the second half of NLS 1 (PAP(H1-493)) had a more severe effect. Similar results were obtained using the specific polyadenylation activity assay. Deletion of the first 16 non-conserved amino acids of hsPAP did not influence the specific or nonspecific activity of hsPAP (Table II). We conclude that hsPAP and hsPAPII have similar organization of functional domains.

View this table: [in this window] [in a new window]   Table II Deletion analysis of PAP The reaction rates were measured in a linear range of protein amount and time. The activity of PAP(H1-736) has arbitrary been set to 100%.

Inhibition of hsPAP Activity by U1A-- The inhibition of PAPII by two molecules of U1A protein is well documented (31, 32). The inhibition requires the last 18 amino acids of PAPII and a region of U1A corresponding to amino acids 102-115. Because hsPAP contains a putative U1A interaction motif, we tested whether it could be inhibited by U1A. Fig. 4 shows that hsPAP was inhibited by an U1A di-peptide but not by the U1A mono-peptide. hsPAPII was inhibited to the same extent under these conditions (data not shown). In these experiments C-terminally tagged recombinant PAP(1-736C) was used, because even a loss of two to three amino acids from the C terminus abolishes the inhibition effect.³ Thus, the C-terminally located U1A interaction motif is functional, and hsPAP activity can be inhibited by U1A as previously reported for bovine PAPII.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Inhibition of hsPAP by U1A. Nonspecific polyadenylation activity assays were performed as detailed under "Experimental Procedures." 0.2 µM primer oligoA15 and 125 fmol of PAP(1-736C) were incubated in the absence or presence of 9.6 pmol of U1A di-peptide or 9.6 pmol of U1A mono-peptide as indicated. The incorporation of [32P]AMP (micromoles/min·mg) is shown.

Subcellular Localization of hsPAP-- Biochemical fractionation studies suggested that the 90-kDa isoform of PAP was nuclear, whereas the 106- and100-kDa isoforms were both nuclear and cytoplasmic (16). To study whether hsPAP localize to the nucleus we used antibodies 20:14 and anti-PAP in a dual staining approach using indirect immunofluorescence techniques. Fig. 5E shows that the monoclonal antibody 20:14 gave a nuclear and weak cytoplasmic staining, as previously reported (36). In the same cell, native hsPAP exhibited an exclusive nuclear distribution (compare panels E and F, Fig. 5). The polyclonal sera specific for exon 22 of hsPAPII type (anti-PAPIIex22) stained both cytoplasm and nucleus (Fig. 5C). Control antibody Y12, recognizing the Sm epitope of general nuclear splicing factors, showed an expected nuclear distribution (Fig. 5A). Intriguingly, in dual staining experiments using anti-PAP and Y12, a high degree of co-localization was observed indicating that hsPAP localizes close to structures enriched in general splicing factors (data not shown). Our data show that endogenous hsPAP is exclusively nuclear, whereas hsPAPII is both nuclear and cytoplasmic.

View larger version (105K): [in this window] [in a new window]   Fig. 5.   Nuclear localization of native hsPAP. Panels A-J: fluorescence of optical sections of HeLa cells immunostained with monoclonal Y12 (A); phase contrast picture of panel A (B); anti-PAPIIex22 (C); phase contrast picture of panel C (D); 20:14 (E); anti-PAP (F); corresponding phase contrast picture of dual labeled cells stained in panels E and F (G). Panels H-J: fluorescence of optical sections of HeLa cells transfected with: H, pPAP(EGFP1-506); I, pPAP(EGFP1-736); J, phase contrast picture of panel I.

The C-terminal Region of hsPAP Is Important for Nuclear Localization-- To identify regions important for guiding hsPAP to the nucleus, we used a transient expression approach with hsPAP/EGFP chimeric proteins. Two C-terminal deletion mutants were constructed: PAP(EGFP1-506) containing the putative NLS 1 region and full-length PAP(EGFP1-736) containing all three putative NLS 1, 2, and 3 regions. Fig. 5 (H-J) shows that PAP(EGFP1-736) was exclusively nuclear, whereas PAP(EGFP1-506) was both nuclear and cytoplasmic. PAP(EGFP1-736) and PAP(EGFP1-506) have predicted molecular masses of ~115 and 70 kDa, respectively. This makes both proteins too large to passively enter the nucleus (47). Thus, we conclude that NLS 1 can mediate partial nuclear import and that the entire C-terminal region (amino acids 506-735) of hsPAP is required for exclusive nuclear localization. We note that this region contains the putative NLS 2 and 3 elements.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Multiple Isoforms of PAP-- Multiple forms of PAP are present in mammalian cell lines and tissues (11, 13, 14, 16, 20, 21). In HeLa cell nuclear extracts three isoforms, having apparent molecular masses of 90, 100, and 106 kDa, have been found (16). In this study, we show that the 90-kDa isoform (hsPAP) is encoded by a distinct locus named PAPOLG. Similar sequences and exon topology of the hsPAP and hsPAPII genes suggest that the two genes arose by gene duplication (Fig. 1A). In summary, at least three mechanisms can encounter for multiple isoforms of PAP, gene duplication, alternative RNA processing, and post-translational modifications (11, 13, 15-17, 20-22). These phenomena unexpectedly increase the diversity of PAP and provoke questions about the functional significance of multiple PAPs in vivo.

hsPAP Is a Bona Fide PAP-- Several lines of evidence suggest that hsPAP is a bona fide PAP. Most importantly, hsPAP is specific for the ATP substrate (Fig. 3A), shows hexanucleotide-dependent polyadenylation activity in the presence of CPSF (Fig. 3B), and has similar kinetic parameters as human/bovine PAPII (Table I and Refs. 24, 29). Furthermore, the amino acid sequence of the region required for PAPII catalytic activity and CPSF/hexanucleotide-dependent polyadenylation activity is highly conserved in hsPAP, suggesting similar functional and structural properties. The resolved crystal structure of PAP (43, 48) demonstrated that amino acids 365 to 513 of bovine PAP, folds into a compact globular domain topologically similar to the RNA binding domains of several RNA binding proteins. The same region of hsPAP contains most likely a similar RNA binding domain and is, in analogy to bovine PAPII, important for CPSF/hexanucleotide-dependent activity (Tables I and II).

hsPAP has been implicated as an enzyme responsible for monoadenylation of small RNAs (14). However, the monoadenylating activity is, in contrast to the polyadenylation activity of PAPII (31, 32) and hsPAP (Fig. 4), not inhibited by U1A (49). The reason for this inability of inhibition is not known yet. Possibly, the U1A inhibitory effect does not occur unless multiple adenosine residues are incorporated by PAP. Another possibility is that an alternatively processed isoform of hsPAP, lacking exon 22, is responsible for the more specialized monoadenylating function in vivo.

hsPAP Is a Nuclear PAP-- In this study we show that hsPAP (i.e. 90-kDa isoform) resides exclusively in the nucleus, whereas the 100- and 106-kDa isoforms of PAP are both nuclear and cytoplasmic (Fig. 5), in keeping with our previous cell fractionation studies (16). It has been reported that polyadenylation factors and a subset of PAP are concentrated at sites of RNA synthesis and associated with domains enriched in splicing factors (36, 50). The antibody used in these studies was the monoclonal 20:14, which recognizes both hsPAP and hsPAP. It is tempting to speculate that the subset of PAP at sites of RNA synthesis and 3'-end processing is hsPAP, because we have observed a high degree of co-localization of hsPAP with basal splicing factors (data not shown). These observations suggest that hsPAP participates in the nuclear polyadenylation reaction. In support of this we have previously shown that a fraction enriched in hsPAP is active both in pre-mRNA cleavage and poly(A) addition (51).

In Fig. 5 we show that the PAP/EGFP chimeric hsPAP is imported into the nucleus. The molecular mass of this chimera is higher than the size limit for passive diffusion through the nuclear pore. This suggests an active transport mechanism (47, 52). The region important for guiding hsPAP to the nucleus must reside in the C-terminal region, because elimination of it (amino acids 507-736) disturbs the observed nuclear pattern of PAP/EGFP chimeric protein. It has been reported that the NLS 1 and 2 elements are important for efficiently directing bovine PAPI and PAPII to the nucleus using transfection experiments (46). In our experiments the NLS 1 element of hsPAP was needed for partial nuclear localization. A careful inspection of the C-terminal sequence of hsPAP revealed a putative bipartite NLS, spanning amino acids 680-714 (NLS 3, Fig. 1B). Two potential phosphorylation sites can be predicted in the C terminus of hsPAP, upstream and in close vicinity of the putative NLS 3. There is increasing amount of data suggesting that regulated phosphorylation is a mechanism that modulates recognition of NLSs by components of the nuclear import machinery (52). A detailed site-directed mutagenic analysis of hsPAP combined with the fusion of NLS 3 to EGFP constructs would be informative to investigate the interesting possibility that phosphorylation may regulate subcellular distribution of hsPAP.

Phylogenetic Conservation of hsPAP-- PAP is a phylogenetically conserved vertebrate variant of PAP present already in the bony fish branch (Table III). The existence of a goldfish hsPAP orthologue supports the hypothesis that gene duplication was an important event in the evolution of early vertebrates (41, 42). In a newly duplicated gene, mutations are generally selectively neutral because of redundancy of genetic information (41, 42). The rate between degenerative and advantageous mutations can be influenced in the gene's favor, if the probability of forming novel regulatory interactions with other genes that are evolving in parallel occurs. Only once a new function has been acquired, the duplicated paralogue will be retained in the population as an evolutionary change. The unique C-terminal region (amino acids 507-736) of hsPAP could be implicated in directing a new function. It is evident that the evolutionary machinery has selected nucleotide substitutions that will create changes at the amino acid level (Fig. 1A). In this study we have shown that at least one of these selected functions is exclusive nuclear localization for hsPAP.

	ACKNOWLEDGEMENTS

We thank Dr. Hans Johansson for valuable advice in bioinformatic approaches, Anders Aspegren for helpful discussions in the immunocytostaining techniques, Dr. Eileen Bridge for providing the Y12 antibody, and Dr. Sam Gunderson for technical advises and for providing us the U1A dipeptide. We thank Daniel Hägerstrand for help in constructing the EGFP-PAP constructs, Reid Prentice for help in purifying recombinant hsPAP mutants, and Ann-Charlotte Thuresson, Javier Martinez, and Yan-Guo Ren for helpful discussions.

	FOOTNOTES

^* This work was supported by the Swedish Strategic Research Foundation, the European Commission through its Training and Mobility of Researchers program, and funds from Uppsala University.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Tel.: 46-18-471-4908; Fax: 46-18-471-4862; E-mail: anders.virtanen@icm.uu.se.

Published, JBC Papers in Press, June 28, 2001, DOI 10.1074/jbc.M104599200

² C. B. Kyriakopoulou, H. Nordvarg, and A. Virtanen, unpublished results.

³ S. Gunderson, personal communication.

	ABBREVIATIONS

The abbreviations used are: PAP, poly(A) polymerase; CPSF, cleavage and polyadenylation specificity factor; RT-PCR, reverse transcription-polymerase chain reaction; b-MEOH, -mercaptoethanol; DTT, dithiothreitol; PBS, phosphate-buffered saline; PFA, paraformaldehyde; kb, kilobase(s); NLS, nuclear localization signal.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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